The invention relates to a method of particle tracking analysis with the aid of scattered light and to an apparatus for detecting and characterizing particles in liquids of all types of the order of magnitude of nanometers.
Suspensions and emulsions as disperse material systems are often occurring forms of particles in liquids. Their uses range from printer ink through cosmetic emulsions to new materials such as quantum dots. Furthermore, nanobubbles, particles in waters, particles in pharmaceutical administrations and exosomes—i.e. nanoparticles with a messenger function released by body cells—are to be included as particles in liquids. The particles occur as individual originally occurring objects or as accumulations in the form of agglomerates or aggregates. In agglomerates, the individual constituents have a loose connection to one another, while aggregates can only be separated from one another by strong forces, for example by grinding processes. There is a great interest in quantifying the particles in their size and shape, and as an agglomerate or aggregate, and quantitatively detecting mixtures thereof. In the field of imaging methods, this is possible with an optical microscope up to a size of a few hundreds of nm, and with an electron microscope up to a minimum size of a few nm. In no method, however, is it yet possible to distinguish between agglomerates and aggregates. Electron microscopic examination suffers from the elaborate sample preparation, and the duration and cost of the analysis.
In contrast to optical microscopy and electron microscopy, optical scattered light analysis is an indirect measurement method for characterization of the particle size. It is used because particles of less than 1 μm (1000 nm) are not compatible with direct observation because of the diffraction limitation.
In a scattered light microscope, the light scattered by particles is used for localization of the particles and tracking of their movement in a video film. There are two versions of scattered light microscopes: the dark-field scattered light microscope with white light illumination and the dark-field laser scattered light microscope with laser illumination (dealt with below).
By analysis of the translational Brownian diffusion movement of each individual particle and subsequent conversion of the measured diffusion constants for each individual particle into the particle size by the Stokes-Einstein formula, the particle size distribution is derived.
When an electric field is applied to the disperse material system, the electrophoretic migration movement is additionally obtained, and therefrom the electric charge at the particle interface with the surrounding liquid. With the aid for example of the Smoluchowski formula, the measured electrophoretic mobility is converted into the so-called zeta potential. In this regard, there is the following consideration:
Disperse systems are, as is known, to be categorized among thermodynamically unstable systems. The time period over which such dispersions remain stable is of essential importance for usability. One instability very often to be observed results from coagulation of particles, which can lead to irreversible particle size growth, or to full separation between the liquid phase and the particle phase. Several precautions are used to reduce coagulation. One of these is electrostatic stabilization. In this case, use is made of the fact that the approach of particles charged in the same way is made difficult by their electrostatic repulsion. This repulsion is commensurately more efficient when the ionic charge of the particles on their interface with the medium is higher. Of crucial importance for this is the electrostatic particle interface potential “PIP”, in particular the zeta potential often derived from the electrophoretic movement (see above). This potential is regarded as a measure which determines the degree of repulsion between neighboring dispersed particles. It therefore has importance in terms of the stability of disperse systems.
In the scattered light arrangements described above, the sample is externally at rest, and only the particles inside the sample move typically according to their size and shape.                The effect of particles resides inter alia in their order of magnitude. For instance, the brilliance of a color may be dependent inter alia on the size distribution, and the site of the effect of a pharmaceutical administration on the size of the carrier particle, for example of a liposome droplet or of gold particles coated with proteins.        Furthermore, the size of particles gives information about their quality, uniformity and usability. If, for example, there are too many agglomerates of a type of particle (protein) present or other types of substances are admixed, then the usability is called into question.        The particle shape also represents an important discriminating feature. In homogenized milk, for example, the fat droplets are broken up to the size of the casein particles of 300 nm. The difference between the two components consists only in the shape. In conventional size measurement methods of DLS, dynamic light scattering, disk centrifuging and ultrasound spectroscopy, fat droplets and casein cannot be distinguished from one another. The particle shape of particles below a size of about 1 μm has to date been measurable only with the aid of electron microscopy. Albeit only statistically and after enormous sample preparation. Dynamic in-situ observation of the particles in the carrier liquid is not possible.        The uncertainty about the correctness of the result of a size distribution is therefore related in conventional DLS scattered light methods to the fact that the scattered light coming from the scattered light volume is collectively gathered on a single detector element. The fluctuation of the scattered light signal is employed for the size distribution. In this case, it is not possible to distinguish whether the fluctuation is caused by the translational movement of the particles, on which the calculation of the particle size by conversion with the aid of the Stokes-Einstein equation is based, or by the rotation of unshaped particles. This is because the center-of-mass variations of a rotating particle aggregate contribute for example to the collective scattered light signal and lead to a “parasitic” fine component, which however cannot be identified per se. An additional uncertainty occurs in the case of substance mixtures, the scattered light behavior of which is different. In the case of substance mixtures, a misevaluation of the various components therefore occurs. If the particles of the different substances are of equal size, it firstly cannot even be suspected that more than one substance type is present in the sample, and even less which components thereof are present.        In electron microscopy as an imaging method, the shape can be measured. However, agglomerates cannot be distinguished from aggregates.        It is therefore desirable to develop a method which offers pattern recognition in a similar way to an electron microscope, but which is substantially fast and more economical and involves only little risk of sample modification by the preparation of the sample for measurement.        Furthermore, it is desirable to distinguish as many features as possible from discrete analysis of the individual particles, which are tracked by video film. Specifically, during the positional change of the particles, sometimes even out of the microscope focus, the particles adopt a constantly changing orientation with respect to the observing microscope. High-intensity vibrations of the individual particles are then found. All these phenomena are admittedly regarded classically as a difficulty of the particle tracking measurement method. Regarded positively, however, these difficulties offer the immense opportunity of a) distinguishing translation of the particles from rotation and thereby eliminating parasitic fine components and b) deriving a set of additional information from the dynamic behavior of the particles during their passage in the video film. This differs from the other methods such as DLS, electron microscopy and disk centrifuging.        In this invention, dynamic multiparameter analysis is thus used as an advantage in order to obtain even more valuable information than previously possible from the dynamics described.        One great difficulty in the PTA method remains the fact that it is necessary to produce an enormously high light contrast (signal/noise ratio in the video detector) for the analysis of nanoparticles. This is because the light scattering of nanoparticles decreases by more than the 6th power toward smaller diameters. Above all, it is necessary to ensure that the light contrast of the weakly luminous particles with respect to the background is maximal and is not attenuated by scattered light. With one sensitive camera alone, this is not achieved. In a scattered light arrangement, there is always parasitic light due to reflections of the exciting laser light at edges and cell walls, and this light also finds its way somehow into the video camera. Comparison with the optimal black night sky during star observation is obvious. The invention of measures for contrast improvement is used to be able to carry out the dynamic pattern recognition on nanoparticles in a size range which is as wide as possible.        Another difficulty with the PTA measuring technique results from the fact that only a small size measurement range of at most 8 to 1 can be recorded simultaneously with one camera setting. In the case of samples with a wider particle size distribution, up to 3 sample dilution stages a 1:3 to 1:4 with up 3 different camera settings are necessary. Dilution automation combined with an intuitive camera setting would substantially simplify the measurement and make it almost error-free in terms of operation. The additional fitting of a miniature pH probe is a further step in the direction of automation. Most users of PTA involve biochemically medical diagnosis. This involves very small sample quantities and often personnel who have difficulties with new types of analysis methods. In the case of samples with the need to measure the zeta potential, it is important to measure and register the ionic properties of the surrounding liquid. The two important parameters which characterize the ionic state in the vicinity of the particle interface are conductivity and pH. They should jointly be registered automatically and without intervention by personnel.        
DE 10 2008 007 743 B3, in the name of the same Applicant, describes a method and an apparatus which relate to the detection of the particle distribution in liquids.
It is pointed out here that there are various physical methods for measuring the PIP.
In the prior art, reference is made in this regard inter alia to the U.S. Pat. No. 3,764,512 A, which discloses an apparatus for the optical detection of particles of a suspension in a cuvette 14, having the following features:                a) the cuvette is positioned in a defined way by means of a mount,        b) the cuvette is irradiated by means of an optical irradiation device and observed at a right angle to the optical axis of the irradiation device by an observation device,        c) the position of the focus of the irradiation device and the position of the focus of the observation device can respectively be displaced in a motorized fashion over the spatial inner region of the cuvette, or by means of focusing adjustment.        
This apparatus has the disadvantage that the optical arrangement is to be focused manually by manually adapting the two focal positions to one another until the image is seen as sharp.
In order to avoid these disadvantages, in DE 10 2008 007 743 B3 according to patent claim 1, an apparatus for the optical detection of particles of a suspension in a cuvette (1) having the following features is protected:                a) the cuvette is positioned in a defined way by means of a mount,        b) the cuvette is irradiated by means of an optical irradiation device and observed at a right angle to the optical axis of the irradiation device by an observation device,        c) the focus of the irradiation device and the focus of the observation device can be displaced in a motorized fashion over the spatial inner region of the cuvette to an arbitrary point by a control apparatus,        d) an approach of the position of the focus of the irradiation device to the position of the focus of the observation device, or vice versa, for the purpose of focusing at a point is monitored in a detection apparatus and/or represented on a display screen.        
Although this method is universally usable, during the measurement of nanoparticles by scattered light or fluorescent light it is limited by the stray light background.